The invention relates generally to cavitation-assisted methods that are used for processing heterogeneous media and mixtures via formation of cavitation bubbles that represent distinct mini-reactors, and uses the energy released upon implosion of these cavities to modify said fluids. The device and method may find applications in biofuel production, chemical, pharmaceutical, food and other industries.
More particularly, the invention relates to the disintegration of unicellular and/or multicellular algal microorganisms and their intracellular organelles to release oil and other cell contents and recover the ingredients. The invention utilizes the implosion energy of cavities to break either reversibly or irreversibly the cell walls and/or membranes of the microorganisms and organelles to release their biochemical constituents, followed by their separation and subsequent conversion into modified, more valuable products, i.e., algal oil.
Biodiesel is an alternative fuel, as it has been designated by the U.S. Department of Energy and the Department of Transportation, and is registered with the U.S. EPA as a fuel. It is biodegradable ester-based fuel that is used as a substitute for petroleum diesel. In the B20 blend (20% biodiesel and 80% petroleum diesel), biodiesel significantly reduces emissions and the level of toxic contaminants in exhaust. Biodiesel can be used in any diesel engine, without the need for mechanical alterations. In practice, biodiesel is manufactured from vegetable oils, animal fats, greases, or other sources of triglycerides by a catalyst-assisted transesterification of triglycerides with methanol, ethanol or by similar methods. The usage of the flow-through hydrodynamic devices greatly increases the reaction rate, improving the yield and composition of the produced biodiesel.
Algal oil is a valuable agricultural land-saving alternative to plant feedstock, such as soybean oil, canola and palm oil or animal, fish and bird fat and tallow. Algae are the simplest plants that live in a water environment. Many algae are unicellular that may or may not have a cell wall. Similar to other plants, algae are photosynthetic. They utilize carbon dioxide as a carbon source and store energy in the form of lipids within the intracellular oil bodies, surrounded by membranes (Barsanti and Gualtieri, 2006). Algae multiply and grow at a very fast rate and, depending on the genetic background and growth conditions, may have very high oil content. In biodiesel production, the wild type strains of algae or mutants and genetically modified microorganisms, which are designed and selected to produce enhanced levels of oil and/or high levels of oleic acid, are preferred. Such strains can be obtained by PCR mutagenesis or by exposure to ultraviolet or ionizing radiation and chemical mutagens. Oil-overproducing strains can be engineered with the help of the directed evolution and other biotechnological techniques known to those skilled in the art (Glick and Pasternak, 2003). More methods to transform algae are disclosed in U.S. Pat. No. 5,661,017 to Dunahay et al.
The process involves the cultivation of microorganisms capable of producing the algal oils in an appropriately controlled environment, wherein the cells are properly treated to stimulate production of oil. Algae produce substantial amount of triacylglycerols as a storage lipid under photo-oxidative stress, nitrogen depletion or other adverse environmental conditions. The produced oil is preferably enriched in oleic acid. Synthesis and sequestration of oil into cytosolic lipid bodies appear to be a protective mechanism by which algal microorganisms cope with stress conditions. Fatty acids, the building blocks for triglycerides, are synthesized in the chloroplast. Acetyl CoA carboxylase is a key enzyme in regulating the rate of fatty acid synthesis. (Hu et al., 2008)
The U.S. DOE Aquatic Species Program has studied algae for twenty years and reported that the algae species can produce up to 60% of their body weight in the form of triglycerides. The complete report is available at http://www.ott.doe.gov/biofuels/pds/biodiesel from algae ps.pdf. In some species of algae, the oil may account for more than half of the cellular mass, which corresponds to more than 80% of the cell dry weight (Molina et al., 2003) [Molina Grima E, Belarbi E-H, Acién Fernández F G, Robles Medina A, Chisti Y (2003) Recovery of microalgal biomass and metabolites: Process options and economics. Biotechnol. Adv. 20: 491-515]. In comparison to agricultural crops, algae-based technology yields significantly more oil per acre. For example, soybean and rapeseed generate about 50 and 130 gallons of oil per acre, respectively, while algae yields up to 10,000 gallons of oil per acre of the water reservoir. Thus, algae, namely diatoms and green algae, are considered to be a promising sustainable feedstock for the production of oil and biodiesel.
There are a number of methods for disintegration of algae and recovering the algal oil, such as bead-assisted milling, pressing, extraction with organic solvents, enzymatic degradation, lysis using osmosis and ultrasonic and microwave-assisted disruption (Cravotto et al., 2008). In most practical applications, the dried and broken cells are extracted with a suitable solvent, preferably hexane, for about two days. The average consumption of hexane is about 5 liters per kilogram of biomass (U.S. Pat. No. 5,164,308 to Kyle). Typically, the crude green oil contains contaminants, such as diglycerides, chlorophyll and other pigments and sterol esters and should be purified until the light yellow oil composed mainly of triglycerides is obtained.
If desired, the cells can be broken, using ultrasonic techniques. Sonication generates cavitation bubbles that collapse violently during a high-pressure cycle. During the implosion of these cavities, very high pressure and temperature and high-speed jets are produced. The resulting shear forces break the cell walls and intracellular organelle membranes, releasing triglycerides into the surrounding fluid, usually hexane or cyclohexane. After the oil is fully dissolved in the organic solvent, the cell debris is filtered out and the solvent is distilled off to recover the oil. However, the cavitation induced by sound waves in the acoustic range (20 Hz-20 KHz) or the ultrasound range (>20 KHz) does not offer an optimized method for cell disruption. For instance, the intensity threshold of ultrasound cavitation in water is >0.3 W/cm2. The typical power requirements for the ultrasonic device integrated inline is approximately 1000 kW for 20-100 m3/h flows.
U.S. Pat. No. 5,629,185 to Stanzl et al. discloses a process for disintegrating dispersions or suspensions of algal microorganisms using ultrasonication for the purpose of recovering cellular constituents. The selected parameters that include sonotrode immersion angle and length of immersion, ratio of sonotrode immersion relative to the acoustic irradiation volume and the ratio of extent of immersion to the solid content of the medium to be sonicated permit the establishment of a particular geometry form for the acoustic irradiation container and method optimization.
The main disadvantage of the sonication technology is the batch environment. Since the effect of sonication diminishes with an increase in distance from the radiation source, the treatment efficacy depends on the size of the irradiation container and is low with large vessels. Sonication disruption cannot be used efficiently in many continuous processes because the energy density and the required residence time would be insufficient for the high throughput of a continuous process. A residence time of about one hour is typically needed for batch process completion. In addition, the alterations are not uniform and occur at particular locations, depending on the frequency of the radiation and the interference pattern. Thus, the efficacy of the sound disruption is further lowered.
High-shear technologies of cell disruption that are used in large-scale production employ the rotor-stator, valve-type or fixed-geometry processors. In these devices, cell broths are subjected to shear forces that pull cells apart. The rotor-stator method is not suitable for the difficult-to-lyse cells and provides highly variable yields. The valve-type disruptors (French press or pumped-fluid processor) destroy cells by forcing the cell-enriched medium through a valve at a pressure of 20-30 Kpsi or higher. The shear force is regulated by controlling the pressure and valve tension. With cultures requiring multiple treatments, cooling is generally required. In the French press, a hydraulic pump drives a piston within a cylindrical body filled with the sample to squeeze it past a needle valve. Once past the valve, the pressure drops, generating shear forces that destroys cells. The device is not suited for processing large volumes and is hard to clean. The mechanically pumped-fluid processors force the medium at a constant volume flow past a spring-loaded valve and are prone to valve clogging. The fixed-geometry processors disrupt microorganisms and cells by forcing them at a high pressure through a chamber that houses a narrow channel and allows controlled cell breakage without using detergents or salts. The fixed geometry of the chamber provides superior reproducibility, while requiring fewer passes.
It has been found that flow-through hydrodynamic cavitation can disintegrate algal cells, allowing fast and efficient release of all intracellular oil. It is well known that a drastic increase in both pressure and temperature and vigorous mixing provided by either acoustic or hydrodynamic cavitation initiates and accelerates numerous reactions and processes. Enhancing the reaction yields and process outcomes by means of the energy released upon the collapse of cavities in a liquid medium has found numerous applications in synthesis, expedition of chemical reactions, homogenizing and other technologies. Although extreme pressure or tremendous heat can be detrimental, the outcome of the optimized cavitation treatment is often beneficial.
Cavitation can be of many different origins, for example, hydrodynamic, acoustic, laser-induced or can be generated by injecting steam into a sub-cooled fluid, which produces collapse conditions similar to those of hydrodynamic and acoustic cavitations (Young, 1999; Gogate, 2008; Mahulkar et al., 2008). Coupling of two or more cavitation-generating techniques provides even better results. For example, direct steam injection coupled with acoustic cavitation increases treatment efficiency by sixteen times, as compared to acoustic cavitation alone.
When fluid temperature approaches the boiling point, the formation of bubbles becomes noticeable. If fluid is pumped through a hydrodynamic cavitation apparatus with a pump at the proper flow velocity, as a result of the decreased hydrostatic pressure (Bernoulli's principle), vapor filled cavitation bubbles form at a concentration of hundreds in one milliliter. The bubbles take up space normally occupied by fluid resisting the flow and increasing the pressure. The formation and growth of the cavities can be prevented by an increase in pressure. When the vapor-filled cavities relocate to a slow-velocity/high-pressure zone, they collapse within 10−8-10−6 s. The implosion results in a localized sharp increase in both pressure and temperature, i.e., as much as 1,000 atm and 5,000° C. or more, and produces local jet streams with velocities reaching 100 m/s and higher (Suslick, 1989; Didenko et al., 1999; Suslick et al., 1999; Young, 1999). The collapse of the cavities generates shock waves, vigorous shearing forces and heat, and releases a substantial amount of energy, which activates atoms, molecules and radicals located within the bubbles and in the surrounding fluid, and initiates chemical reactions and processes and/or dissipates into the surrounding. In many cases, the implosion is light emission-free. In other cases, it is coincidental with the emission of ultraviolet and/or visible light, which may favour photochemical reactions or generate radicals (Zhang et al., 2008). A side effect of the excessively high pressure is heat release, which may become a problem if overheating is detrimental to product's quality and cavitation device operation safety.
The cavitation phenomenon is categorized by the dimensionless cavitation number Cv, defined as: C=(P−Pv)/0.5ρV2, where P is the recovered pressure downstream of a constriction, Pv is the vapor pressure of the fluid, V is an average velocity of fluid at the constriction, and ρ is the fluid density. The cavitation number, at which the cavitation starts, is defined as the cavitation inception number Cvi. Cavitation ideally begins at Cvi=1. Another important term is the processing ratio, which is a number of cavitation events in a unit of flow. The smaller the bubble, the greater the energy released during its implosion. The effect of surface tension and size of cavities on the hydrostatic pressure is defined as follows: Pi=P0+2a/R, where Pi is the hydrostatic pressure, a is the surface tension, and R is the radius of the bubble (Gogate, 2008; Passandideh-Fard and Roohi, 2008).
The flow-through hydrodynamic cavitation does not require using a batch container, as does sound or ultrasound-induced cavitation. Numerous flow-through hydrodynamic devices are known. See, for example, U.S. Pat. No. 6,705,396 to Ivannikov et al., U.S. Pat. Nos. 7,338,551, 7,207,712, 6502,979, 5,971,601 and 5,969,207 to Kozyuk, which disclose different hydrodynamic cavitation reactors and their applications.
Liquid cell cultures are viscous media, and flow-through hydrodynamic cavitation is very profound in such fluids. If a high-cell-density broth is fed into the cavitation device at a proper velocity, causing a flow pressure to drop below the solvent's vapor pressure, cavitation will occur. The cavitation temporarily separates the high-boiling-point compounds and particles from the entrapped gases, water vapor and the vapors of the affected fluids that can be found in the bubbles. Small particulate and impurities serve as nuclei of the cavitation bubbles that may reach a few millimeters in diameter, depending on the conditions. The implosion of the cavities breaks cells, releases their contents into the surrounding fluid and mixes them with the solvent. The temperature, composition and pH of the medium to be treated can be adjusted to increase the efficiency of the oil recovery. In general, the optimization will depend on the cell structure of the species, the composition of the extracting medium, and the presence of contaminants.
To lower the release of unwanted intracellular constituents, including the cell wall debris or products of the denaturation, it is desirable to control cell breakage. The flow-through hydrodynamic cavitation process has a low residence time and does not require much solvent, reducing the chance of denaturation and decomposition of targeted compounds and valuable by-products, lowering downstream production cost and improving the environmental effect.
Algal cell walls contain glycoproteins, sporopollenin, calcium, cellulose and other polysaccharides that are used for algal taxonomy. The diatoms synthesize their cell walls from orthosilicic acid. The cell membrane that lies under the cell wall is a selectively permeable lipid bilayer, which is found in all cells. Algal microorganisms often exhibit high stability and resistance to degradation by enzymes and strong chemical reagents. Therefore, the disintegration of algae with the controlled flow-through hydrodynamic cavitation process may be combined with the enzymatic, chemical or other lysis to achieve a synergetic effect. Hydrodynamic cavitation assists protease, lipase, cellulase, amylase, lysozyme, lysostaphin, zymolase, mutanolysin, glycanase, mannase and other enzymes in penetrating and degrading the cell walls and membranes, resulting in an improved extraction of oil with higher yields. The process can be scaled up easily to accommodate large process streams.
At the present time, with the cost of energy rising rapidly, it is highly desirable to shorten processing time and lower energy consumption to secure as large a profit margin as possible. However, the prior art techniques do not offer the most efficient method of algal cell disruption, especially the hard-to-break species, in the shortest amount of time possible. A need, therefore, exists for the advanced flow-through device for processing algae broth with a minimal residence time and energy cost resulting in oil-containing mixtures with improved characteristics that can be easier to purify. The advanced, compact, and efficient apparatus is particularly desired at the algae growth locations and in field conditions, when throughput is a key factor. The present invention provides such a device, while delivering intracellular oil and other valuable constituents and by-products within a short time.